Self-inactivating helper virus for the production of high-capacity adenoviral vectors

Abstract

Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (C) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which C is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers 410 10 infectious units and o0.1% HV contamination. The residual HVs lacked C and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of

Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center

“…No empty particles were produced when part of the packaging sequence was deleted (mentioned in [64]), but an Ad5 construct containing loxP sites flanking the packaging sequence does accumulate empty particles when propagated in Cre-expressing cells [27, 28]. This observation does not rule out the concerted model, because deficient Cre function may result in incomplete cleavage of Ψ, still allowing an initial interaction between capsid and core components; also, it has been observed in the development of helper systems for gutless AdV production that residual helper virus can package genomes lacking Ψ [76]. Systematic molecular and structural characterization of assembly products from the various mutants would be required to assess this point.…”

Safety and antitumor effect of oncolytic and helper-dependent adenoviruses expressing interleukin-12 variants in a hamster pancreatic cancer model

“…Production and amplification of this vector has been previously described. 40 The ratio i.u. per viral particles of OAV-scIL-12, OAV-scIL-12-miR, OAV-scIL-12-GPI, OAV-scIL-12-TM, Ad-WT-Luc and HC-Ad/RUmIL-12 batches used in this study were 136, 136, 94, 129, 81 and 39, respectively.…”

Liver Gene Therapy Approaches for Acute Intermittent Porphyria: Metabolic Correction and Immunological Hurdles

“…57 Good manufacturing practice (GMP) production of this vector is complicated, although significant advances have occurred based on improvements in the helper adenovirus, which can be more easily and efficiently removed during high scale manufacture. 58 Johansson et al 59 developed a recombinant first-generation adenovirus vector in which the expression of the human housekeeping PBGD was controlled by the strong constitutive human cytomegalovirus promoter. The intravenous administration of this vector resulted in a higher hepatic PBGD expression 7 days after vector administration.…”